Electrochemical potentometric sensing

ABSTRACT

The invention relates to a method of determining a charged particle concentration in an analyte ( 100 ), the method comprising steps of: i) determining at least two measurement points of a surface-potential versus interface-temperature curve (c 1,  c 2,  c 3,  c 4 ), wherein the interface temperature is defined as a temperature of the interface between a measurement electrode and the analyte ( 100 ), wherein the surface-potential is defined at the interface, and ii) calculating the charged particle concentration from locations of the at least two measurement points of said curve (c 1,  c 2,  c 3,  c 4 ).This method, which still is a potentiometric electrochemical measurement, exploits the temperature dependency of a surface-potential of a measurement electrode. The invention further provides an electrochemical sensor and electrochemical sensor system for determining a charged particle concentration in an analyte. The invention also provides various sensors which can be used to determine the charged particle concentration, i.e. EGFET&#39;s and EIS capacitors.

FIELD OF THE INVENTION

The invention relates to an electrochemical sensor for determining acharged particle concentration in an analyte, a semiconductor devicecomprising such sensor, an RF-ID tag comprising such sensor, and to anelectrochemical sensor system for determining a charged particleconcentration in an analyte. The invention further relates to a methodof determining a charged particle concentration in an analyte.

BACKGROUND OF THE INVENTION

The pH-value is an integral parameter of every (aqueous) solution. Itdescribes to which degree the solution is alkaline or acidic. Over awide range it is well approximated by: pH=−log [H⁺], wherein [H⁺]denotes the hydrogen ion concentration of the solution in mol/L.Measuring a pH-value of an aqueous solution is a routine task in theindustry and also in laboratories for process control and analysis.However, it could also become interesting for a wider range ofapplications if the pH-measurement units (sensor plus electronics)become sufficiently inexpensive. For example, there is a large potentialfor pH-measurement to monitor the quality of (liquid) perishables in thesupply chain or even at the customer's himself. Experimental techniquesfor measuring ion concentrations (in particular pH) can be divided intotwo classes, non-electrochemical methods, e.g. optical (indicator dyes),catalytic, and swelling of polymers (gels), and electrochemical methods.The latter are widely used for many applications in industry andlaboratories. Electrochemical ion concentration sensors rely on thepotentiometric principle, i.e. they measure the electrical potential φat a solid/liquid interface or across a membrane which is a function ofthe ion concentration to be determined. φ can be calculated from theNernst equation: φ=kT/(nq) In(a₁/a₂), wherein k is the Boltzmannconstant, T the absolute temperature in Kelvin, q the elementary charge,n the ionic charge (e.g., n=1 for H₃O⁺, Na⁺; n=2 for Ca²⁺), and a₁,a₂the respective activities at both sides of the membrane/interface.

Ion concentrations at both sides of the membrane/interface (1 and 2) arerepresented in terms of activities a_(i)=f_(i)*c_(i) with f_(i) beingthe respective activity coefficient (f_(i)=1 for diluted electrolytes)and c_(i) the respective ion concentration. According to the Nernstequation the electrode potential is a logarithmic function of the ionactivity on one side of the membrane/interface if the activity on theother side is kept constant. Depending on the type of ion described by“a”, the sensor is sensitive to H₃O⁺-ions, Na⁺-ions, Ca²⁺-ions, etc.

All major pH (ion) measurement electrodes operate according to theprinciple described above, including the well-known glass electrodes(different glass compositions have been developed that are sensitive topH, pNa, pK, etc., respectively), antimony electrodes, ISFET's (IonSensitive Field Effect Transistors) and EIS capacitors (ElectrolyteInsulator Semiconductor capacitors; here the flat-band voltage is afunction of the pH/pNa/pK/etc of the electrolyte).

In order to measure a potential difference, i.e. voltage, a referenceelectrode is needed; for the ISFETS and EIS devices the referenceelectrode also defines the electrolyte potential to set the operatingpoint or do a capacitance voltage (C-V) measurement. The potential ofthe reference electrode with respect to the electrolyte potential mustremain constant irrespective of the electrolyte composition. Besides thestandard hydrogen electrode the Ag/AgCl electrode is the most well-knownreference electrode. It consists of a chlorinated silver wire in contactwith a well defined electrolyte (often 3 mol/L KCl). Galvanic contactbetween the analyte and the electrolyte is established via a diaphragm,such as a porous frit from glass or ceramics. During operation theelectrolyte must continuously flow out of the reference electrode intothe analyte. Other reference electrodes, e.g. calomel (based on mercury)or Tl/TlCl electrodes, are used for specific applications, e.g. atelevated temperatures. Their principle is the same as for the Ag/AgClelectrode, in particular with respect to the use of liquid electrolyteand contact via a diaphragm.

The problem with the known electrochemical sensors is that they requirean accurate reference electrode with a reference analyte in order todetermine the charged particle concentration from a measured potential(difference). Using reference electrodes, and in particular accuratereference electrodes, involves all kinds of difficulties such as thefollowing:

-   -   Electrolyte outflow in a reference electrode through the        diaphragm is essential. That means the electrolyte needs to be        refilled regularly. Moreover, the pressure conditions must be        such that the outflow is guaranteed, i.e. the pressure in the        analyte cannot be higher than in the reference electrode        (otherwise the analyte enters the reference electrode and        changes its potential, which is called reference electrode        poisoning);    -   Clogging of the diaphragm of the reference electrode causes        measurement errors (depending on the application regular        cleaning is needed);    -   Most reference electrodes have rather large dimensions, which        makes it difficult/impossible to integrate them into a        miniaturized device. Some miniature reference electrodes exist        but they have a limited lifetime (because reference electrolyte        cannot be refilled);    -   Reference electrodes have a limited temperature range, e.g., for        high temperatures a Tl/TlCl electrode must be used; and    -   Some reference electrodes may react to other environmental        parameters, for example, the silver in Ag/AgCl electrodes is        light sensitive.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an electrochemical sensorfor determining a charged particle concentration, which does not requirea conventional reference electrode which produces a known potential.

The invention is defined by the independent claims. The dependent claimsdefine advantageous embodiments.

In a first aspect, the invention relates to an electrochemical sensorfor determining a charged particle concentration in an analyte, thesensor comprising:

-   -   a measurement electrode for measuring a surface-potential at an        interface between the measurement electrode and the analyte in        which the measurement electrode is immersed in operational use,        and    -   a control means for measuring the surface-potential at least two        different temperatures of the interface to obtain at least two        measurement points of a surface-potential versus        interface-temperature curve.

The effect of the features of the electrochemical sensor in accordancewith the invention is as follows. In all electrochemical sensors, thereactions of interest occur at the surface of the measurement electrode.It is of interest to measure the potential across the interface betweenthe surface of the measurement electrode and the solution (i.e., thesurface potential). However, it is impossible to control or measure thissurface potential without placing another electrode in the solution.Thus, two potentials must be considered, neither of which can bemeasured independently. The reason why in the electrochemical sensorsknown from the prior art the reference electrode must produce a fairlyaccurate reference voltage is that otherwise the charged particleconcentration cannot be determined from the Nernst equation, i.e. theabsolute value of the surface-potential must be known.

The inventors have realized that the charged particle concentration mayalso be determined in a different manner, namely it may be determinedfrom the surface-potential versus temperature curve, and in particularfrom the slope of this curve. In order to do so the electrochemicalsensor comprises a measurement electrode for measuring asurface-potential at an interface of the measurement electrode and theanalyte in which the measurement electrode is immersed in operationaluse. The electrochemical sensor further comprises a control means formeasuring the surface-potential at at least two different temperaturesof the interface between the measurement electrode and the analyte toobtain at least two measurement points of a surface-potential versusinterface-temperature curve.

The electrochemical sensor enables determination of the charged particleconcentration in the analyte as follows. First, the control meansensures that the temperature of the interface between the measurementelectrode and the analyte reaches a first value. Subsequently, themeasurement electrode can be “read-out” to give the surface-potentialcorresponding with the first temperature. These two steps aresubsequently repeated for at least one other temperature different fromthe first temperature, which gives a total of at least two measurementpoints of a surface-potential versus temperature curve and which enablesto determine a corresponding slope. The absolute values of thecorresponding potential of the at least two measurement points in saidcurve are dependent on the absolute potential of the analyte as definedby the reference electrode. However, it is not required that thereference interface-potential is known and accurately determined, i.e.that it does not vary with the charged particle concentration, becausethe charged particle concentration is determined by the slope of saidcurve. Once the slope has been determined, the correspondingcharged-particle concentration can be calculated from the slope. Forthis purpose a pseudo-reference electrode is sufficient. Apseudo-reference electrode is so named because it does not maintain aconstant potential (potential depends on analyte composition);therefore, by definition, it is not a true/real reference electrode.However, its potential depends on conditions in a well-defined manner;if the conditions are known, the potential can be calculated and theelectrode can be used as for reference potential.

In an embodiment of the sensor in accordance with the invention thecontrol means comprises a temperature setting means arranged for settinga temperature of the interface at at least two different temperatures ofthe interface. Providing such temperature setting means is a first wayof enabling to measure the surface-potential at at least two differenttemperatures of the interface between the measurement electrode and theanalyte to obtain at least two measurement points of said curve.

In an embodiment of the sensor in accordance with the invention thetemperature setting means comprises a heater and/or a cooler. Using atleast one of these two components enables to set a temperature of theinterface between the measurement electrode and the analyte. An exampleof a temperature settings means which can be used for heating and/orcooling is a peltier element.

In an embodiment of the sensor in accordance with the invention thetemperature setting means comprises a resistive heater wherein thetemperature is set by controlling a current through the heater. Theadvantage of the sensor in accordance with the invention is that theabsolute value of the temperatures at which the surface-potential ismeasured need not be known. For obtaining slope information in saidcurve, it is only required to know the temperature shift. Thisembodiment is particularly advantageous because, in an environment,having a certain temperature and a constant heat loss, i.e. anenvironment in thermal equilibrium, each respective current value (orcurrent duty cycle in case of pulsed current) through the resistiveheater will correspond with a predetermined steady-state temperature ofthe analyte at the interface. Expressed differently, the currentcontrols the interface temperature shift with respect to theenvironmental temperature, which gives the required information fordetermining the charged particle concentration. No additionaltemperature sensor for determining the absolute interface temperature isrequired.

In an embodiment of the sensor in accordance with the invention thetemperature setting means further comprises means for determining thepower dissipation of the resistive heater and thereby determining thetemperature of the interface between the measurement electrode and theanalyte.

In an embodiment of the sensor in accordance with the invention thecontrol means comprises a controller, the controller being coupled tothe measurement electrode and being arranged for initiating themeasuring of the surface-potential with the measurement electrode at atleast two different temperatures of the interface. Providing suchcontroller, is a second way of enabling to measure the surface-potentialat at least two different temperatures of the interface between themeasurement electrode and the analyte to obtain at least two measurementpoints of said curve.

In an embodiment of the sensor in accordance with the invention thecontroller comprises a temperature sensor for measuring the temperatureof the interface, and the controller is further arranged for initiatingthe measuring of the surface-potential at a desired interfacetemperature value. This embodiment is advantageous in case thetemperature of the analyte is not constant over time, i.e. because ofexternal influences. All what is required in that situation, is that thecontroller initiates the measurement of the surface-potential at twodifferent temperature values measured by the temperature sensor.

In an embodiment of the sensor in accordance with the invention thecontroller comprises storage means for storing the respective measuredvalues of the surface-potential and optionally the respective values ofthe temperature of the interface. Surface-potentials (optionallytogether with the respective temperature or temperature change) thathave been stored in the storage means, can be read-out at any time inorder to enable calculation of the charged particle concentration.

In an embodiment of the sensor in accordance with the invention thecontroller comprises a processor unit for calculating the chargedparticle concentration from the at least two measurement points of saidcurve. This embodiment conveniently provides the charged particleconcentration when the measurement has been carried out. There is noneed to do this manually anymore. The processor unit may be undersoftware control or it may be a universal piece of hardware such as agate array.

In an embodiment of the sensor in accordance with the invention themeasurement electrode is selected from a group comprising: aglass-electrode, an ion-sensitive field-effect transistor, anion-sensitive extended gate field-effect transistor, and an electrolytesemiconductor insulator structure. This list comprises the mostconvenient measurement electrodes.

In an embodiment of the sensor in accordance with the invention themeasurement electrode comprises the ion-sensitive extended gatefield-effect transistor which comprises i) a field-effect transistor,ii) a sensor electrode being electrically coupled to a gate of thefield-effect transistor, and iii) an ion-sensitive sensor dielectricprovided on the sensor electrode, the sensor electrode being arrangedfor contacting the analyte via the ion-sensitive sensor dielectric inoperational use, and

the temperature setting means comprises a resistive heater which isarranged in thermal coupling with the sensor electrode for setting atemperature of the interface between the sensor dielectric and theanalyte in operational use. In this embodiment the conventionalextended-gate field effect transistor (EGFET) is modified. It has beenprovided with a temperature setting means, in the form of a resistiveheater, which is arranged for setting the temperature of the interfacein operational use. This embodiment of the sensor constitutes a relativesimple but very effective EGFET-based sensor for carrying out thecharged particle measurement in accordance with the invention. Thisembodiment of the sensor can be easily integrated in a semiconductordevice. The fact that this sensor does not need an accurate conventionalreference electrode further improves the miniaturization of the sensor.A simple pseudo-reference electrode (a simple electrode wire or metalcontact in the analyte) is sufficient.

In a variation on last mentioned embodiment the sensor dielectric may beprovided with or exchanged with an ion-exchange resin. Ion-exchangeresins are based on special organic polymer membranes which contain aspecific ion-exchange substance (resin). This is the most widespreadtype of ion-specific electrode. Usage of specific resins allowspreparation of selective electrodes for tens of different ions, bothsingle-atom or multi-atom. They are also the most widespread electrodeswith anionic selectivity. However, such electrodes have low chemical andphysical durability as well as “survival time”. An example is thepotassium selective electrode, based on valinomycin as an ion-exchangeagent.

In an embodiment of the sensor in accordance with the invention themeasurement electrode comprises the electrolyte semiconductor insulatorstructure which comprises i) a conductive contact layer, ii) asemiconductor layer provided on the contact layer, iii) an ion-sensitivesensor dielectric provided on the semiconductor layer, the semiconductorlayer being arranged for contacting the analyte via the ion-sensitivesensor dielectric in operational use, and the temperature setting meanscomprises a resistive heater which is arranged in thermal coupling withthe electrolyte semiconductor insulator structure for setting atemperature of the interface between the sensor dielectric and theanalyte in operational use. In this embodiment the conventionalelectrolyte semiconductor insulator

(EIS) capacitor is modified. It has been provided with a temperaturesetting means, in the form of a resistive heater, which is arranged forsetting the temperature of the interface in operational use. Thisembodiment of the sensor constitutes a relative simple but veryeffective EIS-based sensor for carrying out the charged particlemeasurement in accordance with the invention. This embodiment of thesensor can be easily integrated in a semiconductor device. The fact thatthis sensor does not need an accurate conventional reference electrodefurther improves the miniaturization of the sensor. A simplepseudo-reference electrode (a simple electrode wire or metal contact inthe analyte) is sufficient. In EIS-based sensors the flat-band voltageof the EIS capacitor yields information on the pH/ion concentration ofthe electrolyte. It is determined by C-V (capacitance-voltage)measurements or with a constant-capacitance method. Both methods requirea reference electrode and an electrode to modulate the analyte potentialfor the capacitance measurements. If only small modulation currents arerequired, the modulation can also be done with the reference electrodeitself. Otherwise a potentiostat and 3-electrode configuration areneeded. Again the temperature at the dielectric/electrolyte interface ismodulated with a heater underneath the EIS layer stack. Temperaturechanges affect the surface potential causing a shift in the flat-bandvoltage. Thus the surface potential is indirectly measured via theflat-band voltage. The semiconductor layer may comprises semiconductormaterials, such as silicon, germanium, silicon-germanium, III-Vcompounds, II-VI compounds, etc.

In a variation on last mentioned embodiment the sensor dielectric may beprovided with or exchanged with an ion-exchange resin. Ion-exchangeresins are based on special organic polymer membranes which contain aspecific ion-exchange substance (resin). This is the most widespreadtype of ion-specific electrode. Usage of specific resins allowspreparation of selective electrodes for tens of different ions, bothsingle-atom or multi-atom. They are also the most widespread electrodeswith anionic selectivity. However, such electrodes have low chemical andphysical durability as well as “survival time”. An example is thepotassium selective electrode, based on valinomycin as an ion-exchangeagent.

In an embodiment of the sensor in accordance with the invention thesensor is arranged for determining a hydrogen ion concentration andthereby a pH-value of the analyte.

In an embodiment of the sensor in accordance with the invention thesensor dielectric is further provided with a probe molecule layercomprising probe molecules, such as i) antibodies, and ii) DNA / RNAstrands, the probe molecule layer being in direct contact with theanalyte in operational use, the sensor dielectric therebybeing-configured for binding charged target molecules for enabling todetermine a charged target molecule concentration in the analyte. Thisembodiment of the sensor constitutes a molecule sensor, which makes useof the same principle as the charged particle sensor in accordance withthe invention (measurement at two different temperatures). Such moleculesensor has a wide application area. In a variation on this embodimentthe probe molecules are directly provided on the electrode. In thatembodiment the sensor dielectric is not required.

In an embodiment of the sensor in accordance with the invention thecharged target molecules are charged biomolecules.

There are various application areas for molecule or biosensors, forexample: drug discovery, DNA sequencing, disease detection at thehospital/doctor (point of care), tumor marking, home use (e.g. glucose),security (biological warfare agents), forensic research. Correspondingbiomolecules that may be of interest in these areas are: drugs, DNA,viruses and pathogens, tumor markers, glucose, antibodies, etc.

In an embodiment of the sensor in accordance with the invention thesensor further comprises a pseudo-reference electrode for providing areference potential to the analyte, the reference potential beingdefined at a further interface of the pseudo-reference electrode and theanalyte in which the pseudo-reference electrode is immersed inoperational use. As the sensor in accordance with the invention onlyneeds a pseudo-reference electrode, this reference electrode may beadvantageously integrated with the measurement electrode.

In a second aspect, the invention relates to a semiconductor devicecomprising an electrochemical sensor in accordance with the invention.It is a great advantage of the invention that the electrochemical sensorcan be integrated into a semiconductor device. All mentioned features inthe embodiments can be integrated onto the same semiconductor device,including the temperature setting means, the control means, thecontroller, the temperature sensor, the pseudo-reference electrode, dataprocessing means, memory etc.

In a third aspect, the invention relates to an RFID-tag comprising anelectrochemical sensor in accordance with the invention. The inventionis advantageously applied in this application area.

In a fourth aspect, the invention relates to electrochemical sensorsystem for determining a charged particle concentration in an analytecomprising:

-   -   a measurement electrode for measuring a surface-potential at an        interface between a measurement electrode and the analyte in        which the measurement electrode is immersed in operational use;    -   a temperature setting means arranged for setting a temperature        of the interface at at least two different temperatures, and    -   a controller coupled to the measurement electrode and being        arranged for initiating the measuring of the surface-potential        with the measurement electrode at at least two different        temperatures of the interface.

The advantages and effects of the electrochemical sensor system inaccordance with the invention follow that of the electrochemical sensor.All embodiments of the electrochemical sensor apply mutatis mutandis tothe electrochemical sensor system.

In a fifth aspect, the invention relates to method of determining acharged particle concentration in an analyte, the method comprisingsteps of:

-   -   determining at least two measurement points of a        surface-potential versus interface-temperature curve, wherein        the interface temperature is defined as a temperature of the        interface between a measurement electrode and the analyte,        wherein the surface-potential is defined at the interface;    -   calculating the charged particle concentration from locations of        the at least two measurement points of said curve.

The advantages and effects of the method in accordance with theinvention follow that of corresponding embodiments of theelectrochemical sensor. The inventors have realized that the particleconcentration information is hidden in the slope of thesurface-potential versus interface-temperature curve. A vertical shiftof said curve does not have any influence on the slope. Thus anyconstant potential offset caused by an “inaccuracy” of the referenceelectrode does not have an effect on the measured slope and consequentlythe determined charged particle concentration. Expressed differently, ifthe reference electrode produces an output voltage, which is unknownupfront (i.e., because its output value depends on a yet-undeterminedcharged particle concentration), the invention still works. In themethod of the prior art, the absolute value of the surface-potential hasto be known for calculating the charged particle concentration.Therefore, in the prior art, a reference electrode is required whichcreates a potential, which is known and independent from the chargedparticle (ion) concentration of the analyte (that is, these electrodesensure a constant analyte potential). The method of the invention doesnot require such reference electrode with a defined and independentoutput potential. A pseudo- or quasi reference electrode, which outputdepends on the charged particle concentration (and thus produces anoutput value which is unknown upfront) is sufficient. Any type ofreference electrode (that is chemically inert to the analyte) will do.The only function the reference electrode has to perform is theestablishment of a galvanic contact to the analyte such that theelectric circuit is closed.

In an embodiment of the method in accordance with the invention the stepof calculating the charged particle concentration comprises thefollowing sub-steps: i) deriving the slope from the at least twomeasurement points, and ii) calculating the charged particleconcentration in the analyte from the slope.

In an embodiment of the method in accordance with the invention, in thestep of determining, at least three measurement points of said curve aredetermined, and wherein the step of calculating the charged particleconcentration comprises the following sub-steps: i) determining astraight fitting line using the at least three measurement points ofsaid curve, and ii) calculating the charged particle concentration fromthe straight fitting line. The advantage of this embodiment of themethod is that measurement noise and measurement errors are reduced.

In an embodiment of the method in accordance with the invention thesub-step of calculating the charged particle concentration comprises: a)determining a slope of the straight fitting line, and b) calculating thecharged-particle concentration from the slope.

In an embodiment of the method in accordance with the invention the stepof determining of said curve comprises sub-steps of:

-   -   setting the interface temperature to a first value;    -   determining a first value of the surface-potential at the        interface, wherein the first value of the interface temperature        and the first value of the surface-potential together define a        first respective one of the measurement points of said curve;    -   setting the temperature of the interface to a second value        different from the first value, and    -   determining a second value of the surface-potential at the        interface, wherein the second value of the interface temperature        and the second value of the surface-potential together define a        second respective one of the at least two measurement points of        said curve.

This embodiment of the method constitutes a possible implementation ofdetermining said curve.

In an embodiment of the method in accordance with the invention thedifference between the first value of the temperature of the interfaceand the second value of the temperature of the interface is smaller thana predefined threshold, preferably smaller than or equal to 10K, andeven more preferably smaller than or equal to 5K. Keeping thetemperature difference between the first and second measurement within acertain threshold ensures that a measurement error, which is the resultof a temperature dependency of a specific parameter of the sensor, isreduced. This applies especially in case of a dielectric sensor layerwhen the temperature dependence of the sensitivity parameter a isunknown. Keeping a small temperature range also reduces the powerconsumption. The pH of the analyte may itself be temperature dependent(e.g. buffers have temperature dependent buffer capacity). Thus if thepH of a solution must be known at a certain temperature the measurementprocess should not deviate too much from this temperature itself. One ofthose temperature dependent parameters is the sensitivity of thedielectric of the ISFET-based measurement electrode, which parameter isknown to be temperature dependent. What is considered as an acceptablemeasurement error generally depends on the application. In case of a pHmeasurements an error of 0.1 pH is acceptable for most applications.

In an embodiment of the method in accordance with the invention the stepof determining at least two measurement points of said curve is done bydetermining respective values of an output quantity that is indicativeof the surface potential. Depending on the chosen type of transducer itmay be that the output is a quantity that is representative of thesurface-potential, i.e. a current through a transistor (for example anISFET or EGFET).

It is important to note that, despite the fact that a real referenceelectrode is no longer needed in the electrochemical sensor inaccordance with the invention, a real reference electrode may still beapplied in the measurement principle in accordance with the invention(i.e. determining a charged-particle concentration from the slope of apotential-versus-temperature curve).

These and other aspects of the invention are apparent from and will beelucidated with reference to the embodiments described hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 shows some formula's for explaining the potentiometricmeasurement principle as known from the prior art;

FIGS. 2( a) to 2(c) show conventional electrodes and referenceelectrodes known from the prior art;

FIG. 3 shows some formula's for explaining the potentiometricmeasurement principle in accordance with the invention;

FIG. 4 shows a diagram with a couple of potential difference versusinterface-temperature change curves for different charged particleconcentrations;

FIGS. 5( a) and 5(b) show two embodiments of the electrochemical sensorin accordance with an embodiment of the invention;

FIGS. 6( a) to 6(d) show four different sensor-heater arrangements inaccordance with other embodiments of the invention, and

FIGS. 7( a) to 7(d) show the manufacturing and operation principle of anelectrochemical biosensor in accordance with yet another embodiment ofthe invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention provides a new method for determining a charged particle,i.e. ions and charged biomolecules, concentration in a liquid analyte.In an embodiment the method concerns determination of a hydrogen ionconcentration and thereby the pH-value. It is based on measurements atdifferent temperatures with a suitable electrochemical sensor. Theelectrochemical sensor may comprise measurement electrodes, such asglass electrodes, ISFETs, and EIS capacitors. The charged particleconcentration is calculated from the measurement electrode readings andthe respective temperatures. Because of the new measurement principle noaccurate reference electrodes are needed any more. All problems andissues associated with these reference electrodes are thereby prevented(e.g. maintenance and refill of electrolyte, bulky device, limitationsin temperature range, etc.). Moreover, the new method of measuring acharge particle concentration is a dynamic measurement type (thetemperature is modulated), which minimizes drift and reduces the needfor frequent calibration.

In view of the above the invention provides a method of determining acharged particle concentration, an electrochemical sensor for enablingto determine the charged particle concentration using such method, anelectrochemical sensor system for enabling to determine the chargedparticle concentration.

In order to facilitate the discussion of the detailed embodiments a fewexpressions are defined hereinafter.

Throughout this description the term “interface temperature” should beinterpreted as the temperature of a volume around the interface whichincludes volume with electrode material and a volume with analyte.

In electrochemistry, the Nernst equation is an equation which can beused (in conjunction with other information) to determine theequilibrium reduction potential of a half-cell in an electrochemicalcell.

A half cell is a structure that contains a conductive electrode and asurrounding conductive electrolyte separated by a naturally-occurringHelmholtz double layer. Chemical reactions within this layer momentarilypump electric charges between the electrode and the electrolyte,resulting in a potential difference between the electrode and theelectrolyte. The typical reaction involves a metal atom in the electrodebeing dissolved and transported as a positive ion across the doublelayer, causing the electrolyte to acquire a net positive charge whilethe electrode acquires a net negative charge. The growing potentialdifference creates an intense electric field within the double layer,and the potential rises in value until the field halts the netcharge-pumping reactions. In a similar way the Nernst equation alsodescribes the surface potential at the interface of a dielectric and anelectrolyte or across a membrane with different ion concentrations inthe electrolytes on either side.

Throughout this description the term “reference electrode” refers to anelectrode which has a stable and well-known electrode potential. Thehigh stability of the electrode potential is usually reached byemploying a redox system with constant (buffered or saturated)concentrations of each participants of the redox reaction. Referenceelectrodes are used to build an electrochemical cell in conjunction withan electrode the potential of which is to be determined. Each electroderepresents a half cell; both are required to complete the circuit andmeasure the unknown potential.

Throughout this description the term “pseudo-reference electrode” refersto an reference electrode which does not maintain a constant potential.By definition, a pseudo-reference electrode is not a true referenceelectrode. However, its potential depends on conditions in awell-defined manner; if the conditions are known, the potential can becalculated and the electrode can be used as for reference potential.

Throughout this description the term “measurement electrode” isconsidered either a glass electrode, ISFET or an EIS capacitor.

Throughout this description the term “charged particle” refers to ionsand charged bio-molecules.

Throughout this description the term “interconnect layer” should beconsidered as synonym to “metallization layer” or “metal layer”. Bothterms are used interchangeably and have to be interpreted as the layercomprising conductors (any conducting material), the insulating layer inwhich the conductors are embedded, and any vias (=contacts) tounderlying layers. These terms are well-known to the person skilled inthe art of semiconductor technology.

Throughout this description the term “substrate” should be interpretedbroadly. The substrate may comprise an active layer with elements, suchas transistors and diodes, which form the components of an electroniccircuit. The substrate may further comprise interconnections between theelements which may be laid out in one or more interconnect layers andmay further contain passive elements such as capacitors, resistors andinductors. In the figures, the elements have been left out in order tofacilitate the understanding of the invention. The active layer in whichthe elements are formed may also be called a semiconductor body. Thesemiconductor body may comprise any one of the following semiconductormaterials and compositions like silicon (Si), germanium (Ge), silicongermanium (SiGe), gallium-arsenide (GaAs) and other III-V compounds likeindium-phosphide (InP), cadmium sulfide (CdS) and other II-VI compounds,or combinations of these materials and compositions as well assemiconducting polymers. The active elements together may form anelectronic circuit. In any case, connection of the active elements isdone via interconnect layers. These interconnect layers have parasiticcapacitances which are defined by the dielectric constant of surroundingmaterials. The semiconductor body may even comprise contacts to lowerlayers (e.g. diffusion regions at the surface of an active region).

FIG. 1 shows some formula's for explaining the potentiometricmeasurement principle as known from the prior art. In the description ofthe figures the main principle will be explained with measurement of aconcentration of hydrogen ions (pH-value). However, it must be stressedthat the invention is also applicable to any other kind of chargedparticle concentration, i.e., Na⁺-ions, K⁺-ions, Ca²⁺-ions, etc.

The pH-value is an integral parameter of every (aqueous) solution. Itdescribes to which degree the solution is alkaline or acidic. Over awide range it is well approximated by: pH=−log [H⁺], wherein [H⁺]denotes the proton concentration of the solution in mol/L.pH-measurement is a routine task in industry and also in laboratoriesfor process control and analysis. However, it could also becomeinteresting for a wider application range if the pH-measurement units(sensor plus electronics) become sufficiently inexpensive. E.g., thereis a large potential for pH-measurement to monitor the quality of(liquid) perishables in the supply chain or even at the customerhimself. Experimental techniques for measuring ion concentrations (as isthe case in pH-measurements) can be divided into two classes,non-electrochemical methods, e.g., optical (indicator dyes), catalytic,and swelling of polymers (gels), and electrochemical methods. The latterare widely used for many applications in industry and laboratories.Electrochemical ion concentration sensors rely on the potentiometricprinciple, i.e. they measure the electrical potential φ across asolid/liquid interface which is a function of the ion concentration tobe determined. The potential φ can be calculated from the Nernstequation, given in formula (1) of FIG. 1. In this formula k is theBoltzmann constant, T the absolute temperature in Kelvin, q theelementary charge, and n the ionic charge (e.g. n=1 for H₃O⁺, Na⁺; n=2for Ca²⁺). Ion concentrations at both sides of the membrane/interface (1and 2) are represented in terms of activities a_(i)=f_(i)*c_(i) withf_(i) being the activity coefficient (f_(i)=1 for diluted electrolytes)and c_(i) the respective ion concentration in mol/L. According to theNernst equation the electrode potential is a logarithmic function of theion activity on one side of the membrane/interface if the activity onthe other side is kept constant. Depending on the type of ion describedby parameter “α” the sensor is sensitive to H₃O⁺-ions, Na⁺-ions,Ca²⁺-ions, etc.

FIGS. 2( a) to 2(c) show conventional electrodes and referenceelectrodes known from the prior art. All major pH-(ion)-measurementelectrodes work according to the principle described above, includingthe well-known glass electrodes (different glass compositions sensitiveto pH, pNa, pK etc. have been developed), antimony electrodes, ISFET's(Ion Sensitive Filed Effect Transistor) and EIS capacitors (ElectrolyteInsulator Semiconductor capacitor; here the flat-band voltage is afunction of the pH of the electrolyte). It is not possible to measure apotential; but it is possible to measure potential differences. In anycase, in order to measure a potential difference with a measurementelectrode a reference electrode is needed, wherein the potentialdifference is generated by a difference in the measurement electrodepotential φ_(m) and the reference electrode potential φ_(ref) (seeformula (2) in FIG. 1). In the case of ISFET and EIS devices asmeasurement electrode the reference electrode is also used to set theoperating point and close the electric loop. In the prior art, thepotential of the reference electrode φ_(ref) with respect to theelectrolyte potential must remain constant irrespective of the analytecomposition. Thus, in the prior art, what is measured is the potentialdifference Δφbetween the measurement electrode potential φ_(m) and thereference electrode potential φ_(ref). This is given by formula (2) inFIG. 1.

In the case of a pH-measurement with a glass-electrode and aconventional reference electrode (with a reference liquid), thepotential difference can be given by (3a) formula in FIG. 1. In formula(3a) pHin stands for the pH of the electrolyte in the glass-electrodeand pHout stands for the pH of the analyte (which has to be determined).In fact formula (3) is the sum of two surface potentials at the insideand outside of the glass electrode as well as the contact potential ofthe wire inside the glass electrode with the electrolyte in theglass-electrode φ_(cont) and the reference electrode potential φ_(ref).However, in this configuration these terms cancel each other out whenboth electrodes have the same temperature. The derivation of formula(3a) and more information on reference electrodes can be found in thefollowing publication:

-   -   “Measuring, modeling, and controlling the PH-value and the        dynamic chemical state.” By Jean-Peter Ylén, Helsinki University        of Technology, Control Engineering Laboratory, Report 127, Espoo        2001 [REF1]. This document has been incorporated by reference in        its entirety.

In the case of a pH-measurement with an ISFET-measurement electrode anda conventional reference electrode, the potential difference can begiven by (3b) formula in FIG. 1. In formula (3b) pHpzc stands for thepoint of zero charge of the ISFET-measurement electrode (a materialproperty defined by the dielectric sensor layer of the ISFET) and pHoutstands for the pH of the analyte (which has to be determined). Thederivation of formula (3b) and more information on ISFET electrodes canbe found in the following publication:

-   -   P. Bergveld, “Thirty years of ISFETOLOGY. What happened in the        past 30 years and what may happen in the next 30 years.”,        Sensors and Actuators B 88 (2003) 1-20 [REF2]. This document has        been incorporated by reference in its entirety.

Besides the standard hydrogen electrode, the Ag/AgCl electrode is themost well-known reference electrode. This reference electrode RE isillustrated in FIG. 2( a). It consists of a chlorinated silver wire 10(Ag/AgCl) in contact with a well-defined electrolyte 20 (often 3 mol/LKCl). Galvanic contact to the analyte is established via a diaphragm 30(porous frit from glass or ceramics, etc.). During operation theelectrolyte 20 must continuously flow out of the reference electrode REinto the analyte. Other reference electrodes, e.g. calomel electrodes(based on mercury) or Tl/TlCl electrodes are used for specificapplications, e.g. at elevated temperatures. Their principle is the sameas for the Ag/AgCl electrode, in particular the use of a liquidelectrolyte 20 and contact via a diaphragm 30. The chlorinated silverwire 10 is connected to a contact cable 40.

FIG. 2( b) illustrates a measurement set-up in which the referenceelectrode RE is used in combination with a glass electrode GE. Bothelectrodes GE, RE are immersed into the analyte 100 in operational use.The glass electrode GE comprises a chlorinated silver wire 10 (Ag/AgCl)in contact with an electrolyte 20′ (buffer solution) with a well-definedpH_(in)-value. The electrolyte 20′ is provided in a pH-sensitive glassmembrane 31, which is produced from a special glass. Its thickness isusually between 50-200 μm, but in the measurement of very aggressivesolutions it can be even 1 mm. After immersion in water the glasselectrode can measure the process solution 100 (analyte). A potentialdifference between the analyte 100 and the glass surface is created, andthis difference is a function of the activity of H₃O⁺-ions and thus alsoa function of the pH-value of the analyte 100. The chlorinated silverwire 10′ is connected to a further contact cable 40′. The cable 40 andthe further cable 40′ are both connect to the input of a voltmeter VM.The voltmeter gives the potential difference Δφ as given by formula (3a)in FIG. 1. More information about glass electrodes can be found in thefirst reference (REF1) given in this description.

FIG. 2( c) illustrates a measurement set-up in which the referenceelectrode RE is used in combination with an ISFET measurement electrodeIE. Both electrodes IE, RE are immersed into the analyte in operationaluse. The ISFET measurement electrode IE comprises a transistorstructure, which is very similar to a conventional field-effecttransistor (FET). It comprises a p-type substrate 5 having an n-typesource Src and an n-type drain Drn provided at a surface thereofdefining a channel region in between. A gate dielectric 32 is providedon the substrate 5 covering source Src, drain Drn and channel.Alternative a p-type transistor can be used. A main difference is thatthe gate dielectric 32 is in direct contact with the analyte 100 insteadof with a poly/metal gate contact. The gate dielectric 32 is the ion/pHsensitive layer (in an example embodiment it comprises SiO₂, but otherdielectrics, such as Ta₂O₅ can also be used). The transistor acts astransducer that converts the potential difference into a current betweenthe source Src and drain Drn of the transistor. Above the channel regionthe dielectric may be thinner than elsewhere, in order to increase thesensitivity of the ISFET (better inversion of the channel in case of apredefined surface potential generation at the dielectric layer 32).More information about ISFET's can be found in the second reference(REF2) given in this description. A reference electrode RE is providedin the analyte 100 in order to establish a “working point” (referencepotential) for the ISFET and define the analyte potential. A potentialset by this reference electrode RE may be considered as the gate voltageV_(G) of a conventional field-effect transistor. In the prior artpH-measurements it is of utmost importance that the potential of thereference electrode is independent of the composition of the analyte.

FIG. 3 shows some formula's for explaining the potentiometricmeasurement principle in accordance with the invention. An essentialfeature of the invention is to execute potentiometric pH/ionmeasurements at different temperatures in the (same) analyte. Whiletemperature changes must be compensated or taken into account with theconventional potentiometric measurement principle of the prior art theinvention exploits the temperature dependency of the sensor output todetermine the quantity to be measured, e.g., the pH-value or ionconcentration of a solution (or a charged biomolecule concentration aswill be discussed in FIG. 7). The arguments described hereafter relateto pH-value but also apply to ion concentration or charged biomoleculeconcentration, then the pH needs to be replaced by pK and the chargenumber n must be taken into account).

The potential difference equation for a combination of a glass electrodeand a conventional reference electrode (with reference liquid) isrepeated in formula (4a) in FIG. 3, wherein pHout denotes the pH-valueat the outside (analyte) and pHin denotes the pH-value of theelectrolyte inside (ln10≈2.3). The inventors have realized that formula(4a) can be looked at differently. According to this formula Δφ shows alinear dependence on T with the slope of the straight line m given byformula (4b) in FIG. 3. It must be noted that all parameters of thisformula are known or fixed, except for pHout which is the pH-value ofthe analyte to be measured. Following this approach the pH-value of ananalyte can be obtained by recording the potential difference Δφ atdifferent temperatures T, determining the slope m of the Δφ-T curve andsubsequently calculating the pH-value using formula (4c) in FIG. 3.

FIG. 4 shows a diagram with a couple of potential difference (betweenmeasurement electrode and reference electrode) versusinterface-temperature change curves for different charged particleconcentrations. The diagram shows Δφ-ΔT-curves for various pHout-valuesin a temperature range 0K-10K (pHin=7). These curves have a directrelation with the surface-potential versus interface-temperature curves.The slopes of the curves allow a clear discrimination of the differentpH-values. Four curves c1, c2, c3, c4 illustrate a pH-value equal to 5,6, 7, and 8, respectively. Which curve runs horizontal depends on thevalue of parameter “pHin”. In principle parameter “pHout” can becalculated without calibrating the sensor since all parameters informula (4a) in FIG. 3 are known. In reality calibration may still beadvisory because of components of the system that do not behave ideallyand may be temperature dependent (e.g. electrode contacting thereference electrolyte inside a glass electrode with pHin).

It should be noted that being a potential φ cannot be measured alone butonly as a potential difference (voltage) to another potential i.e. thepotential from the reference electrode. However, this does not imply anyrestriction if the reference potential (and all other potentialsinvolved; e.g. potential between the inner electrode and referenceelectrolyte of a glass electrode) is independent of the analytecomposition and only the surface potential of the measurement ismodulated by a very localized change in the interface temperature(temperature at other interfaces e.g. between reference electrode andanalyte are kept constant during the measurement).

Since the information about the pH-value (hydrogen-ion concentration) isconveyed in the slope of the Δφ-ΔT-curves rather than in the absolutevalue of φ (as is the case with conventional potentiometricmeasurements) any vertical shift of the curves has no effect on themeasurement. Thus any potential offset caused by using apseudo-reference electrode instead of a ‘real’ reference electrode isneglected. A pseudo-reference electrode consists of a simple metal wire(e.g. Pt or Ag) immersed in the analyte (sample solution). Thispseudo-reference electrode provides a constant reference potentialduring the measurement, but depends on the analyte composition (e.g. itsion concentration). However, such a pseudo reference electrode is fullysufficient for our invention. For a precise measurement it must only bemade sure that the potential of the pseudo-reference electrode remainsconstant during the measurement itself, i.e. during changing of thetemperature T and recording of the respective values from themeasurement electrode. For the method it is not even necessary to knowthe absolute temperature. The only value which must be known (inarbitrary units) is the interval ΔT between different measurements. Forexample the temperature T can be given as: T=T₀+a*U₂/R*t , whereinparameter “T₀” denotes the temperature at t=0s, parameter “R” denotesthe ohmic resistance of a resistive heater, parameter “U” denotes theapplied voltage and parameter “t” denotes the time the heater isactivated. Parameter “a” comprises all other system parameters e.g. thevolume of the heated liquid and its heat capacity. Substituting thisformula for the temperature with formula (4a) in FIG. 3, gives a formulafor the potential difference Δφ as a function of time t for which theheater is activated. The absolute value of the start temperature T₀ doesnot need to be known, since it only causes a vertical shift of thecurve, whereas the pH-value (pHout) is conveyed in the slope. Acalibration of the system (i.e. measure the slope of a curve with abuffer of defined pHout) may be necessary in order to determineparameter “α”. Moreover, parameter “α” should preferably remain constantbetween calibration and real measurement since it directly affects theslope.

Potentiometric measurements as known from the prior art are staticmeasurements, which rely on the thermodynamic equilibrium. Staticmeasurements are often subject to drift which makes frequent calibrationnecessary. Besides the associated effort and cost, some systems aredifficult to calibrate, e.g. because the sensor is fixed in avessel/pipe and would need to be removed or because the system cannot beaccessed at all (perishable monitoring, medical applications). Drift isa particular problem for ISFET sensors. Various algorithms andprocedures have been developed to predict drift and correct themeasurements Moreover, new sensors must equilibrate for a certain timebefore they can be used. An advantage of the measurement principle ofthe invention is that due to the dynamic measurement principle drift isconsiderably reduced increasing the calibration intervals andmeasurement accuracy. More information on drift and counter-measures canbe found in the following publication:

-   -   S. Jamsab, “An Analytical Technique for Counteracting drift in        Ion-Selective Field effect Transistors (ISFETs)”, IEEE Sensors        J., 4 (6), 795-801, 2004 [REF3]. This document has been        incorporated by reference in its entirety.

Another advantage of the new measurement principle in accordance withthe invention is the noise reduction. If the slope of a Δφ-ΔT-curve isdetermined by fitting a straight line to several φ values recorded atdifferent temperatures, noise and statistical measurement errors areaveraged out.

Until now, for the sake of clarity only the fundamental principles andequations have been shown and discussed. In real applications it mightbe slightly more complex. This also depends on the type of measurementelectrode and reference electrodes chosen.

If a glass electrode is used for the measurements not only the membranepotential across the ion sensitive glass membrane has to be taken intoaccount but also the potentials of the (pseudo) reference andmeasurement electrode (contacting the reference liquid with pHin).Unfortunately, the potential of the reference electrode may also betemperature dependent. However if the same electrode material andelectrolyte are used in the measurement and ‘real’ reference electrodeand both electrodes are kept at the same temperature they cancel out(φ_(cont)→φ_(ref)). This is not the case if a pseudo-reference electrodeis used. In order to prevent errors with this system it is better toheat the analyte only locally near the ion sensitive glass membranewhile the analyte at the pseudo-reference electrode remains at itsinitial temperature.

In the case of a pH-measurement with an ISFET-measurement electrode anda reference electrode, the potential difference can be given by (5a)formula in FIG. 3 wherein the first part describes the surface potential(which yields the information on the pH-value of the analyte) of thedielectric/analyte interface, wherein parameter pHpzc denotes thepoint-of-zero-charge, i.e. the pH-value of the analyte for which theoxide surface is electrically neutral, wherein parameter pHout denotesthe actual pH-value of the analyte in contact with the dielectric,wherein parameter a denotes a temperature dependent sensitivityparameter which is characteristic for the specific ISFET sensordielectric. Parameter α lies between 0 and 1 (in case of a sensitivityequal to 1 the sensor has the maximum sensitivity). Formula's (5b) and(5c) can be derived from formula (5a) in a way that is similar to thatof formula's (4b) and (4c) in FIG. 3.

Parameter a for an ISFET is known to be defined as given in formula (6)in FIG. 3, wherein parameter C_(S) denotes the double layer capacitance(which depends on the ion concentration in the analyte), and whereinparameter β_(S) denotes the surface buffer capacity which is a materialparameter of the sensor dielectric. Other parameters are alreadyexplained earlier in the description.

The temperature dependency of the sensor sensitivity α may complicatethe measurement method a bit. It can be addressed in several ways (orcombinations thereof).

1) Use a sensor dielectric material with high surface buffer capacityβ_(S). This measure minimizes the temperature dependence of thesensitivity α. The advantage of this approach is that the measurementprinciple described above can applied without modification. In apreferred embodiment the sensor dielectric material comprises tantalumoxide (Ta₂O₅) which has the advantage that it has a very high β_(S).

2) Perform the different temperature measurements in a small temperature“window”, e.g. 5K. Within this temperature window the sensitivity α maybe assumed to be constant. Consequently, a small change in thesensitivity α results in a relatively small error and can be neglected.This second approach requires that the calibration and “real”measurement are done at the same temperature. Otherwise the error willincrease because of the earlier mentioned temperature dependency, whichthus results in different slopes.

3) Determine C_(S) and β_(S) during sensor calibration. A singlecalibration run with one reference solution is sufficient. However, thepotential difference must be measured for several temperatures to allowfitting of the Δφ-T-curve in order to obtain C_(S) and β_(S). This isthe most accurate approach but the absolute temperature must be known. Atemperature sensor for determining the absolute temperature is thusrequired.

The method for measuring pH or ion concentrations can be realized byinstalling a small heater/cooler next to the sensor (glass electrode,ISFET). The heater/cooler heats/cools the analyte in close proximity tothe sensor. The sensor readings (representing Δ_(φ)) at differenttemperatures (T measured with integrated sensor or determined fromheating energy) are stored or plotted; the chemical parameter is thenobtained from the slope of the curve according to the method describedabove. Instead of a close-by heater/cooler the analyte temperature canalso be controlled by a remote device and applied to the sensor by afluidic system (e.g. flush liquid onto sensor).

If no temperature sensor is used in the method, sufficient time mustpass between subsequent heat pulses to allow cooling of the sensor tothe initial (ambient) temperature. If only short heat pulses are used aheat wave will propagate towards the dielectric/analyte interfaceleading to a transient temperature increase. Continuous measurement ofthe surface potential (transducer output) will result in amaximum/minimum value, which value shall be used for further dataextraction (when this value is reached the temperature at the interfaceis highest/lowest before it cools off / heats up again). To increasemeasurement accuracy a curve can be fitted to determine the extremevalue (taking into account the temporal behavior of the temperature atthe interface following a heat pulse). A simpler way is to average a fewvalues in an interval around the extreme value.

Where in this specification the wording “obtaining of measurement pointsof a surface-potential versus temperature curve” is used, it is oftenmeant that measurement points of a potential-difference (between thefirst electrode and a (pseudo)-reference electrode) versus temperature(of the interface at the measurement electrode) is meant. Nevertheless,as in the invention it is not required to know the absolute temperature,but only to determine the slope of the surface-potential versustemperature curve, the latter curve has a clear relation with the firstcurve and is sufficient to obtain the slope.

So far, the description of the figures mainly dealt with the method ofdetermining a charged particle concentration in an analyte in accordancewith the invention. However, the invention also relates to anelectrochemical sensor, which can be used to carry out this method. Ithas already been discussed that such electrochemical sensor may compriseconventional measurement electrodes, such as glass electrodes, andconventional reference electrodes. So, in any case the electrochemicalsensor in accordance with the invention must comprise a measurementelectrode for measuring a surface-potential at an interface between themeasurement electrode and the analyte in which the measurement electrodeis immersed in operational use. Further the electrochemical sensor inaccordance with the invention must also comprise at least a controlmeans for enabling to measure the surface-potential at at least twodifferent temperatures of the interface to obtain at least twomeasurement points of a surface-potential versus interface-temperaturecurve. Such control means can be a temperature setting means arrangedfor setting a temperature of the interface at at least two differenttemperatures of the interface. Alternatively, such control means can bea controller, wherein the controller is coupled to the measurementelectrode and is arranged for initiating the measuring of thesurface-potential with the measurement electrode at at least twodifferent temperatures of the interface. A combination of both is alsopossible.

Miniaturized solutions for the electrochemical sensor are of particularinterest as that opens up new application possibilities (due to smallform factor and reduced cost). An example of such miniaturization is theISFET measurement electrode. A disadvantage of the

ISFET is that with the measurement principle of the prior art still anaccurate reference electrode (with reference electrolyte) is required,which electrode cannot be easily miniaturized. Miniaturized versions,which have been reported in the prior art so far, have a very limitedlife-time.

A major advantage of the invention is that this cumbersome referenceelectrode is no longer required. Instead a pseudo-reference electrode(which is basically a metal contact immersed in and in electricalcontact with the analyte in operational use) can be used. This referenceelectrode can be easily integrated into the ISFET using the interconnecttechnology already present. Miniaturization has thus become very easy.Nevertheless, it is still possible to combine the electrochemical sensorof the invention with a conventional reference electrode. As alreadymentioned reference electrode allows to set a DC-potential of theanalyte to a known value, which may be advantageous if the measurementmethod of the invention is combined with conventional measurementmethods.

The main building blocks of an electrochemical sensor in accordance withan embodiment of the invention are:

-   -   a sensor electrode covered with a suitable sensor material        (depending on the application pH or ion sensitive);    -   a heater/cooler in close proximity the sensor, and    -   a transducer for transducing the sensor output into an        electrical signal for further processing.

Moreover, the electrochemical sensor may include circuits for dataprocessing and storage, power supply. The electrochemical sensor mayfurther comprise circuit blocks such as AD/DA converters, digital signalprocessors, memory and RF units for wireless data transfer.

In a first embodiment of the electrochemical sensor the measurementelectrode is an ISFET (as illustrated in FIG. 2( c)). The ISFET has beendiscussed earlier in this description. In order to use an ISFETaccording to our invention a small temperature setting means (i.e. aheater) is needed near the gate dielectric. This could be a resistiveheater (thin lines of metal wire) processed next to or surrounding thegate area, e.g. by metal deposition and etch with a suitable mask. Theheater may be covered by dielectric layers (e.g. be integrated into themetal interconnect) protecting it from direct contact with theelectrolyte. The reference electrode can be used in the analyte to setthe working point of the ISFET. If the ISFET is used according to ourinvention a simple pseudo-reference electrode can be used. A majordisadvantage of ISFETs is the direct contact between analyte and theion-sensitive gate dielectric. This makes CMOS process integrationdifficult because all layers above the sensor gate must be removed (e.g.by etch) to allow direct contact with the analyte. Moreover, because ofthe close contact between analyte and active layer (only thin dielectriclayers for protection) there is a high risk that ions diffuse into theintegrated circuit and shift the threshold voltage of transistors thatare close to the opening and destroy the CMOS circuit.

FIGS. 5( a) and 5(b) show two embodiments of the electrochemical sensorin accordance with an embodiment of the invention. FIG. 5( a) shows aso-called Extended Gate Field-Effect-Transistor (EGFET). FIG. 5( b)shows a so-called Electrolyte Semiconductor

Insulator (EIS) structure.

Referring to FIG. 5( a), in this structure the issues, described in thelast paragraph above, do not exist. It consists of a conventionaltransistor NM having a source Src, a drain Drn, and a gate Gt, e.g. anNMOS transistor. The gate Gt of the transistor NM is connected to asensor electrode Snse via standard metal interconnect ‘wires’. On thesensor electrode Snse a sensor dielectric Snsd is provided that issensitive to certain ions. The sensor has been exemplified in asimplified way to facilitate understanding of the invention. A heaterHtr (temperature setting means) has been provided close, for exampleunderneath, to the sensor electrode Snse and sensor dielectric Snsd.What is important is that the heater Htr is provided such that it isthermally coupled to the sensor part for setting its temperature.

Many variations are possible in this respect. Some of these variationsare illustrated in FIG. 6. The transistor NM of the sensor has afloating gate, because the connection between gate Gt and sensorelectrode Snse is not galvanically connected to any voltage source.Instead, it is surrounded by insulators such as the gate dielectric,sensor dielectric Snsd and interconnect dielectric. The working point ofthe sensor is controlled by a reference electrode, here apseudo-reference electrode PR, in the analyte. The pseudo-referenceelectrode PR can be integrated with the EGFET, for example in the topmetal layer.

The major advantage of the EGFET as compared to the ISFET is that thesensor electrode Snse is implemented in the top metal layer of the chipand thus ‘far away’ from the layer comprising the transistor NM. Thisreduces risk of contamination with, e.g.

alkaline ions, such as Na⁺. Moreover, it allows easy integration withstandard CMOS processes.

Referring to FIG. 5( b), in this structure the issues, described in thebefore-last paragraph, also do not exist, because it can be manufacturedin the upper layer(s) of a chip. The Electrolyte Semiconductor Insulatorstructure comprises a conductive contact layer Cl (e.g. metal pad,silicide) onto which a silicon layer S1 is provided. On the siliconlayer S1 a sensor dielectric Snsd is provided. The stack is similar to aMOS (Metal Oxide Semiconductor) capacitor. It differs from there in thatthe dielectric/oxide is contacted by the analyte rather than by metal.The flat-band voltage of the EIS capacitor yields information on thepH-value/ion concentration of the analyte. It is determined by C-V(capacitance voltage) measurements or with a constant capacitancemethod. Both methods at least require a reference electrode to definethe DC potential of the analyte and to modulate the analyte potentialfor the capacitance measurements. Again the temperature at the sensordielectric/electrolyte interface is modulated with a heater Htr near,for example underneath, the EIS layer stack. Temperature changes affectthe surface potential that subsequently causes a shift in the flat-bandvoltage. Thus the surface potential is indirectly measured via theflat-band voltage. The reference electrode can be a simplepseudo-reference electrode PR for the same reason discussed with FIG. 5(a).

More information on the electrolyte-insulator semiconductor structurecan be found in the following document:

-   -   Shoji Yoshida, Nobuyoshi Hara, and Katsuhisa Sugimoto,        “Development of a Wide Range pH Sensor based on        Electrolyte-Insulator Semiconductor Structure with        Corrosion-Resistant Al₂O₃—Ta₂O₅ and Al₂O₃—ZrO₂ Double-Oxide Thin        Films.”, Journal of The Electrochemical Society, 151 (3)        H53-H58 (2004) [REF4]. This document has been incorporated by        reference in its entirety.

More information on C-V measurements can be found in the followingdocument:

-   -   M. Klein, “CHARACTERIZATION OF ION-SENSITIVE LAYER SYSTEMS WITH        A C(V) MEASUREMENT METHOD OPERATING AT CONSTANT CAPACITANCE.”,        Sensors and Actuators B1 (1-6): p354-356, January 1990 [REFS].        This document has been incorporated by reference in its        entirety.

Because of the special measurement principle of the invention theearlier described problems related to the reference electrode andcalibration are no longer relevant (or at least to a much smallerdegree) for the electrochemical sensor in accordance with the invention.In particular, the embodiments described here can be easily miniaturizedand integrated into standard CMOS devices. Only minor additions to astandard processing scheme are necessary. Moreover, these modificationsare after all conventional processing has been finished, and beforedicing and packaging).

FIGS. 6( a) to 6(d) show four different sensor-heater arrangements inaccordance with other embodiments of the invention. All figures aresimplified, in particular for the sensor. For the sensor only the sensorelectrodes are shown. In FIG. 6( a) the sensor Snsr is arranged as alarge pad, whereas the heater Htr is arranged (in a same plane) aroundthe periphery of the pad. In FIG. 6( b) the heater Htr is arranged underthe sensor pad Snsr in the form of a meander. This configuration ensuresa more uniform temperature of the sensor. In FIG. 6( c) the sensor Snsris arranged as a meander structure, and the heater Htr is arranged, in asame plane, as a meander structure on both sides of the sensor Snsr in ariver-routing fashion. In FIG. 6( d) the sensor Snsr is arranged as ameander structure. The heater Htr is arranged below the sensor Snsr as ameander structure in a 90°-rotated. The actual arrangement of heater Htrand sensor Snsr may considerably affect the temperature uniformity ofthe sensor. The person skilled in the art may easily come up withfurther variations of the sensor Snsr and heater Htr. In any case, whatis important is that the heater Htr (temperature settings means) isthermally coupled to the sensor Snsr for enabling the setting of thesensor temperature.

Method of Manufacturing

Sensor manufacturing of the embodiments of FIGS. 5 and 6 followsstandard CMOS processing schemes. This is the case for the transducersas well as for most parts of the sensor (and heater). If we consider aCMOS process with five metal layers for interconnect the heater can beimplemented as a thin metal line (resistive heater) in Metal4 and thesensor electrode in Metal5 (for the geometries of FIGS. 6( a) and 6(c)both the heater Htr and sensor Snsr can be implemented in Metal5). Metallayers are separated by inter-layer-dielectric (ILD). Standardback-end-of-line processes are used for this, such as (dual)-damasceneprocessing. Depending on the actual interconnect technology aluminum andcopper are the most commonly used metals. The only non-standard stepsare: i) deposition of the sensor dielectric on top of Metal5 and ii)opening of the bond pads. Both steps can be done as the final processingsteps before dicing and packaging. Thus no changes are required for thestandard processing part of the manufacturing method. The sensordielectric can cover the entire device surface (including the heaters inarrangements in FIGS. 6( a) and 6(c)) thus acting as additionalprotective layer against the electrolyte. If a scratchprotection/passivation stack is used the process steps may involve:opening of the scratch protection (lithography, etch) on top of sensorelectrodes and bondpads, uniform deposition of sensor dielectric,removal of sensor dielectric on bond pads.

In order to improve protection, e.g. reduce pin holes, stacks ofdifferent dielectrics can be deposited. The actual sensitivity isdetermined by the final layer in contact with the electrolyte. Fortransducer/sensor configurations that use a pseudo-reference electrodethe dielectric (and scratch protection) is also removed on a metal padin an area later covered by electrolyte to establish a contact(processed together with bondpad opening). If needed, e.g. to improvecorrosion resistance, other metals (silver, gold, platinum etc.) can bedeposited on top of this pad by electrochemical or electrolessdeposition, PVD or CVD etc. with subsequent patterning or lift off.

Energy Consumption

Despite the use of a heater in some embodiment of the electrochemicalsensor in accordance with the invention, the overall energy consumptionis low, because the heated volume can be very small. If we consider asensor having a surface area of 1000 μm2 the overall heat capacity isabout 7.4*10-9 J/K. The following assumptions are made for thiscalculation:

-   -   the heater is assumed in Metal4;    -   the heat propagation to Metal3 and Metal5 are assumed to be        identical;    -   the total thickness of the heated stack is assumed to be around        3.5 μm;    -   aluminum is used as metal;    -   siliconoxide is used as intra-metal-dielectric and inter-layer        dielectric, and;    -   the electrolyte itself needs hardly to be heated only at the        interface to the dielectric).

Doing ten measurements at ten different temperatures with 1K temperaturedifference and cool down in between (starting from the equilibriumtemperature) requires an overall energy of 3.3*10⁻⁷ J which correspondsto 92 pAh (at 1V). This energy is so small that it does not impose anyrestriction to the sensor, even not for miniature sensors in autonomoussensors tags powered with a small battery (capacity in the range of 1mAh). The low-energy consumption is also beneficial for a rapid cooldown. The heated volume is very small (3500 μm³). This means that lessthan 1 μL of analyte is sufficient to act as “reservoir” with constanttemperature. This reservoir serves to cool down the sensor to theinitial temperature after a heat pulse.

As already described earlier in this description, the invention may alsobe applied in different application areas, i.e. in de field of moleculesensors. FIGS. 7( a) to 7(d) show the manufacturing and operationprinciple of an electrochemical biosensor in accordance with yet anotherembodiment of the invention and its principle of operation. Theelectrochemical biosensor is to a large extent very similar to thealready described embodiments of the sensor. Therefore, the biosensorwill only be discussed in as far as it differs from the sensor alreadydescribed. FIG. 7( a) shows such (plain) sensor that has already beendescribed. In FIG. 7( b) the sensor is modified for turning the sensorinto the biosensor. In order to do so the entire surface of the sensordielectric Snsd is provided with a probe molecule layer Pml. Inoperational use the probe molecule layer Pml is in direct contact withthe analyte. The probe molecule layer Pml is applied such that thesensor dielectric is configured for binding charged target molecules inthe analyte. This enables to determine a charged target moleculeconcentration in the analyte. FIGS. 7( c) and 7(d) illustrate theoperation principle of the biosensor. In FIG. 7( c) the sensor isapplied in an analyte having biomolecules Bm in it. Biomolecules Bm thatmatch with functional parts of the probe molecule layer Pml bind to thesurface and change the surface potential of the sensor. In FIG. 7( d)the analyte is replaced by a measuring solution. This step is optional,which depends on which approach, as discussed below, is chosen. Themeasuring solution is an electrolyte that does not contain anybiomolecules but closes the electrical circuit.

The surface of the sensor dielectric Snsd is functionalized with probemolecules capable of binding to target molecules that have to bedetected in the analyte. The functionalized surface may compriseimmobilized nucleic acids, e.g. probe-cDNA or mRNA. When the nucleicacid sequence of the (immobilized) probe-cDNA or mRNA is complementaryto the nucleic acid sequence of the target DNA (in the analyte), theprobe-cDNA or mRNA hybridizes to the DNA fragment and changes the sensorsurface potential. Similarly proteins, hormones and various pathogensmay be detected by immobilizing the respective antibodies on the sensorsurface. Probe-DNA and antibodies may be immobilized using linkers, selfassembled monolayer's (SAM), in situ nucleic acid synthesis, etc. In avariation on this embodiment the probe molecules are directly providedon the first electrode. In that embodiment the first ion-sensitivedielectric is not required.

The core of the measurement principle of the biosensor is the same asfor pH/ion measurement, namely to vary a temperature of the analyte nearthe measurement electrode and measure any change in the potentialdifference, i.e. output from the transducer. However, due to thedifferent “binding”-mechanism of the biomolecules (the binding is notautomatically reversible as is the case for the pH/ion sensor), aslightly modified scheme must be followed. There are multiple approachespossible of which two are discussed below.

Approach 1:

As a first step, a calibration step is performed. In this step ameasurement is done using a reference solution of which its content isknown. The reference solution is an electrolyte with fixed pH and saltconcentration to close the electrical circuit. With “measurement” ismeant a measurement in accordance with the invention at at least twodifferent temperatures (determining at least two different potentialdifferences). The obtained data is saved or stored. This calibrationstep can be done in a manufacturing environment as part of themanufacturing process, if desired.

As a second step, the analyte 100 is applied to the sensor for apredetermined amount of time. During this time period target moleculesare bound to the probe molecule layer Pml.

As a third step, the surface of the sensor is flushed. In this flushingstep in principle any solution can do that does not contain targetmolecules nor remove bound target molecules during flushing.

As a fourth step, another measurement (measurement at at least twodifferent temperatures) is performed on the reference solution. Theresult of this measurement is compared with the data from the referencemeasurement. If target molecules have bound to the probe molecule layerPml they will stay there during flushing and the results from the secondmeasurement will be different form the first. This difference isindicative of the concentration of the target molecules in the analyte100.

Approach 2:

A more simple approach is to measure constantly during application ofthe analyte 100. With “measurement” is again meant measurement at atleast two different temperatures. As the target molecules slowly bind tothe sensor probe molecule layer Pml the sensor readings graduallychange. It is important that the measurements at different temperaturesare performed quickly so that the at least two measurements per“measurement” experience approximately the same biomoleculeconcentration. The difference between measurements right after analyteexposure and measurements after a certain exposure time is indicative ofthe original concentration of target molecules in the analyte 100.

The biosensor may comprise several sensors (e.g. in an array)functionalized with different probe molecules (deposited by ink jetspotting, etc.) to detect different target molecules in a singlemeasurement run.

The thermo potentiometric principle in accordance with the inventiononly allows the detection of charged particles, such as ions, as thesecharged particles attach to the sensor surface and change the surfacepotential (Nernst equation only applies to ions, pH is a special ion:H₃O⁺, OH⁻). Therefore the biosensors in accordance with the inventionare also applicable to charged target molecules. Charged targetmolecules of interest are DNA for example. DNA is known to be charged,although this charge may have many different values. Unlike normal ions,such as Nations, the charge on biomolecules heavily depends on thepH-value of the analyte in which they are dissolved, which makes thesecharged particles somewhat more special.

The invention thus provides a method of determining a charged particlesconcentration in an analyte. This method, which still is apotentiometric electrochemical measurement, exploits the temperaturedependency of a surface-potential of a measurement electrode. Theinvention further provides an electrochemical sensor and electrochemicalsensor system for enabling to determine a charged particle concentrationin an analyte. The invention also provides various sensors which can beused to determine the charged particle concentration, i.e. EGFET's andEIS capacitors.

The invention may be applied in a wide variety of application areas, forexample in ion concentration sensors, and in particular in pH-sensors.Further the invention may be applied in miniature sensors integratedinto autonomous (RFID) tags. The invention may also be applied inpotentiometric sensors with surface modifications, e.g. detection ofbiomolecules attaching to a sensor surface.

Various variations of the sensor and method in accordance with theinvention are possible and do not depart from the scope of the inventionas claimed. These variations for example relate to material choice,layer thickness, spatial arrangement of the sensor parts, etc. Also, inthe method of determining a charged particle concentration in accordancewith an embodiment of the method of the invention, many alterations arepossible. Such alterations fall within the normal routine of the personskilled in the art and do not deviate from the inventive concept heredisclosed. The most important variations are:

-   -   Sensor dielectrics may include materials like: SiO₂, Ta₂O₅, SiN,        TiO₂, HfO₂, Al₂O₃, and similar materials.    -   Non-dielectric sensor materials can also be used, such as        antimony and other metals, polymers, such as Polyaniline,        Polypyrrole, Linear Polyethylenimine, Linear Polypropylenimine,        and similar materials. These materials may be either in direct        contact with the sensor electrode or with a dielectric in        between.

A temperature sensor may be implemented near the sensor to accuratelydetermine the temperature at the interface between sensor material(dielectric) and analyte.

For example, a thermistor can be realized by an additional thin metalwire surrounding the sensor pad (similar to the arrangement of theheater around the pad in FIG. 6( a)).

Several sensors which are configured for different analytes can beimplemented on a single chip, e.g. pH-value and Na⁺-ion concentration.

-   -   An inductive heater may be used instead of a resistive heater.    -   The heater can be operated in constant-power mode (wherein        activation time is adjusted) or in constant-activation-time mode        (wherein the power is adjusted).    -   The sensor capacitance forms a capacitive voltage divider        together with the input capacitance of the transducer (e.g. gate        capacitance of the transistor, input capacitance of operational        amplifier). In order to improve the signal of the sensor, the        sensor capacitance can be increased (it should be preferably        larger than the transducer's input capacitance). The capacitance        can be increased by making a the sensor area larger, or by        making the sensor dielectric layer thinner    -   A peltier element may be used as a cooler (temperature setting        means).    -   The sensor dielectric may be provided with or exchanged with an        ion-exchange resin. Ion-exchange resins are based on special        organic polymer membranes which contain a specific ion-exchange        substance (resin). This is the most widespread type of        ion-specific electrode. Usage of specific resins allows        preparation of selective electrodes for tens of different ions,        both single-atom or multi-atom. They are also the most        widespread electrodes with anionic selectivity. However, such        electrodes have low chemical and physical durability as well as        “survival time”. An example is the potassium selective        electrode, based on valinomycin as an ion-exchange agent.

It should be noted that the above-mentioned embodiments illustraterather than limit the invention, and that those skilled in the art willbe able to design many alternative embodiments without departing fromthe scope of the appended claims. In the claims, any reference signsplaced between parentheses shall not be construed as limiting the claim.Use of the verb “comprise” and its conjugations does not exclude thepresence of elements or steps other than those stated in a claim. Thearticle “a” or “an” preceding an element does not exclude the presenceof a plurality of such elements. The invention may be implemented bymeans of hardware comprising several distinct elements, and by means ofa suitably programmed computer. In the device claim enumerating severalmeans, several of these means may be embodied by one and the same itemof hardware. The mere fact that certain measures are recited in mutuallydifferent dependent claims does not indicate that a combination of thesemeasures cannot be used to advantage. Throughout the Figures, similar orcorresponding features are indicated by same reference numerals orlabels.

1. An electrochemical sensor for determining a charged particle concentration in an analyte, the sensor comprising: a measurement electrode for measuring a surface-potential at an interface between the measurement electrode and the analyte in which the measurement electrode is immersed in operational use, and a control means for measuring the surface-potential at at least two different temperatures of the interface to obtain at least two measurement points of a surface-potential versus interface-temperature curve.
 2. The electrochemical sensor as claimed in claim 1, wherein the control means comprises a temperature setting means (Htr) arranged for setting a temperature of the interface at at least two different temperatures of the interface.
 3. The electrochemical sensor as claimed in claim 1, wherein the control means comprises a controller, the controller being coupled to the measurement electrode and being arranged for initiating the measuring of the surface-potential with the measurement electrode at at least two different temperatures of the interface.
 4. The electrochemical sensor as claimed in claim 3, wherein the controller comprises a temperature sensor for measuring the temperature of the interface, and wherein the controller is further arranged for initiating the measuring of the surface-potential at a desired interface temperature value.
 5. The electrochemical sensor as claimed in claim 3, wherein the controller comprises storage means for storing the respective measured values of the surface-potential and optionally the respective values of the temperature of the interface.
 6. The electrochemical sensor as claimed in claim 2, wherein the measurement electrode comprises an ion-sensitive extended gate field-effect transistor which comprises a field-effect transistor, a sensor electrode being electrically coupled to a gate of the field-effect transistor, and an ion-sensitive sensor dielectric provided on the sensor electrode, the sensor electrode being arranged for contacting the analyte via the ion-sensitive sensor dielectric in operational use, and wherein the temperature setting means comprises a resistive heater which is arranged in thermal coupling with the sensor electrode for setting a temperature of the interface between the sensor dielectric and the analyte in operational use.
 7. The electrochemical sensor as claimed in claim 2, wherein the measurement electrode comprises an electrolyte semiconductor insulator structure which comprises a conductive contact layer, a semiconductor layer provided on the contact layer, an ion-sensitive sensor dielectric provided on the semiconductor layer, the semiconductor layer being arranged for contacting the analyte via the ion-sensitive sensor dielectric in operational use, and wherein the temperature setting means comprises a resistive heater which is arranged in thermal coupling with the electrolyte semiconductor insulator structure for setting a temperature of the interface between the sensor dielectric and the analyte in operational use.
 8. The electrochemical sensor as claimed in claim 6, wherein the sensor dielectric is further provided with a probe molecule layer comprising probe molecules, such as antibodies, and DNA/RNA strands, the probe molecule layer being in direct contact with the analyte in operational use, the sensor dielectric thereby being configured for binding charged target molecules for enabling to determine a charged target molecule concentration in the analyte.
 9. A semiconductor device comprising the electrochemical sensor as claimed in claim
 1. 10. An RF-ID tag comprising the electrochemical sensor as claimed in claim
 1. 11. An electrochemical sensor system for determining a charged particle concentration in an analyte, the system comprising: a measurement electrode for measuring a surface-potential at an interface between a measurement electrode and the analyte in which the measurement electrode is immersed in operational use; a temperature setting means arranged for setting a temperature of the interface at at least two different temperatures, and a controller coupled to the measurement electrode and being arranged for initiating the measuring of the surface-potential with the measurement electrode at at least two different temperatures of the interface.
 12. A method of determining a charged particle concentration in an analyte, the method comprising steps of: determining at least two measurement points of a surface-potential versus interface-temperature curve, wherein the interface temperature is defined as a temperature of the interface between a measurement electrode and the analyte, wherein the surface-potential is defined at the interface; calculating the charged particle concentration from locations of the at least two measurement points of said curve.
 13. The method as claimed in claim 12, wherein the step of determining of said curve comprises sub-steps of: setting the interface temperature to a first value; determining a first value of the surface-potential at the interface, wherein the first value of the interface temperature and the first value of the surface-potential together define a first respective one of the measurement points of said curve; setting the temperature of the interface to a second value different from the first value, and determining a second value of the surface-potential at the interface, wherein the second value of the interface temperature and the second value of the surface-potential together define a second respective one of the at least two measurement points of said curve.
 14. The method as claimed in claim 13, wherein the difference between the first value of the temperature of the interface and the second value of the temperature of the interface is smaller than a predefined threshold. 